Composition containing human umbilical cord blood-derived mesenchymal stem cell for inducing differentiation and proliferation of neural precursor cells or neural stem cells to neural cells

ABSTRACT

A use of a composition comprising umbilical cord blood-derived mesenchymal stem cells for inducing differentiation and proliferation of neural precursor cells or neural stem cells to neural cells is provided, the composition being effective for the treatment of nerve injury diseases.

This is a divisional application of copending application Ser. No.12/516,913, filed on May 29, 2009, which is a national stage applicationunder 35 U.S.C. §371 of PCT Application No. PCT/KR2007/006084, filed onNov. 29, 2007, which claims the benefit of U.S. Patent ProvisionalApplication No. 60/867,875, filed on Nov. 30, 2006, and from KoreanPatent Application No. 10-2006-0120479, filed on Dec. 1, 2006, theentire contents of each of which are incorporated herein by reference intheir entirety.

FIELD OF THE INVENTION

The present invention relates to a use of a composition comprising humanumbilical cord blood-derived mesenchymal stem cell for inducingdifferentiation and proliferation of neural precursor cells or neuralstem cells to neural cells.

BACKGROUND OF THE INVENTION

Stroke, Parkinson's disease, Alzheimer's disease, Pick's disease,Huntington's disease, amyotrophic lateral sclerosis, traumatic centralnervous system disease and spinal cord injury disease involve dysneuriacaused by injury of nerve cells, and they have been generally treated bymedication or surgical operation which may severely damage normal cells.

Recently a cell replacement therapy in which normal cells aretransplanted to replace destroyed or damaged cells has been recognizedto be effective for such diseases, and stem cells, in particular, whichcan be differentiated and proliferated into desired tissues are underintense studies.

Stem cells are unspecialized cells that can be proliferated unlimitedlyin the undifferentiated stage and can be differentiated into diversetissues in response to specific stimuli.

Neural stem cells, from which neurons and/or glia such as astrocytes,oligodendrocytes and/or Schwann cells form, are also undifferentiatedcells having self-reproduction potency. They differentiate into neuralcells, for example neurons or glia via neural precursor cells or gliaprecursor cells.

Mesenchymal stem cells, which differentiate into bone, cartilage,adipose tissue, muscle, tendon, ligament, neural tissue and others, havebeen known to be viable for the cell replacement therapy. Mesenchymalstem cells have been obtained mainly from bone marrow, but suchmesenchymal stem cells provide only limited applications due to theirrestrictive potency for differentiation and proliferation. Further,complicated and often painful operations composed of several steps mustbe conducted for such cell replacement therapy, besides the problem offinding a donor who has histocompatibility antigens identical with thatof a patient to exclude graft versus host reaction during bone marrowtransplantation.

In recent years, the umbilical cord blood has become a target forresearchers because of its high concentrations of stem cells. A numberof trials to treat blood diseases by transplanting umbilical cord bloodto a patient have been conducted, and umbilical cord blood banks, whichpreserve umbilical cord blood in a frozen form until use, have beenestablished for the autologous transplantation therapy.

Unlike the bone marrow, the umbilical cord blood can be obtained by asimple operation from an umbilical cord and it causes little graftversus host reaction. For these reasons, worldwide studies for clinicalapplication of the umbilical cord blood have recently been performed.

The present inventors have also extensively studied umbilical cordblood-derived mesenchymal stem cells and found that they are capable ofinducing differentiation and proliferation of neural precursor cells orneural stem cells to neural cells.

SUMMARY OF THE INVENTION

Therefore, it is an object of the present invention to provide a use ofa composition comprising umbilical cord blood-derived mesenchymal stemcells for inducing differentiation and proliferation of neural precursorcells or neural stem cells to neural cells.

In accordance with another aspect of the present invention, there isprovided a method for inducing differentiation and proliferation ofneural precursor cells or neural stem cells to neural cells, whichcomprises co-culturing umbilical cord blood-derived mesenchymal stemcells with the neural precursor cells or the neural stem cells.

In accordance with a further aspect of the present invention, there isprovided a composition for inducing differentiation and proliferation ofneural precursor cells or neural stem cells to neural cells, comprisingumbilical cord blood-derived mesenchymal stem cells as an activeingredient.

In another aspect of the invention, there is provided a method forproliferating neural precursor cells or neural stem cells, whichincludes bringing the neural precursor cells or neural stem cells to bein contact with umbilical cord blood-derived mesenchymal stem cells. Thecontact may occur in vitro by culturing the neural precursor cells orneural stem cells together with umbilical cord blood-derived mesenchymalstem cells.

According to another aspect of the invention, there is provided a methodfor differentiating neural precursor cells or neural stem cells intoneural cells, which includes bringing the neural precursor cells orneural stem cells to be in contact with umbilical cord blood-derivedmesenchymal stem cells, optionally together with an agent which iscapable of differentiating the neural precursor cells or neural stemcells into neural cells. The agent may be selected from the groupconsisting of ascorbic acid, glia derived neurotrophic factor (GDNF),brain derived neurotrophic factor (BDNF), retinoic acid, insulin andnerve growth factor (NGF).

In another aspect of the inventive embodiment, there is provided amethod for simultaneously proliferating neural precursor cells or neuralstem cells and differentiating the neural precursor cells or the neuralstem cells into neural cells, which includes bringing the neuralprecursor cells or neural stem cells to be in contact with umbilicalcord blood-derived mesenchymal stem cells, optionally together with anagent which is capable of differentiating the neural precursor cells orneural stem cells into neural cells. The contact may occur in vivo byadministering umbilical cord blood-derived mesenchymal stem cells,optionally together with the agent, to a nerve cell injury region of amammal. The contact may occur in vitro by culturing the neural precursorcells or neural stem cells together with umbilical cord blood-derivedmesenchymal stem cells, optionally in the presence of the agent.

According to another aspect of the invention, there is provided a methodfor treating a nerve injury disease, which includes administeringumbilical cord blood-derived mesenchymal stem cells to a nerve cellinjury region of a subject in need of treating the nerve injury disease.The method may further includes administering an agent which is selectedfrom the group consisting of ascorbic acid, glia derived neurotrophicfactor (GDNF), brain derived neurotrophic factor (BDNF), retinoic acid,insulin and nerve growth factor (NGF). The umbilical cord blood-derivedmesenchymal stem cells may be administered as a culture of the umbilicalcord blood-derived mesenchymal stem cells and neural precursor cells ora culture of the umbilical cord blood-derived mesenchymal stem cells andneural stem cells.

In accordance with a still further aspect of the present invention,there is provided a method for treating a nerve injury disease whichcomprises administering the composition to a nerve cell injury region ofa subject in need of treating the nerve injury disease.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects and features of the present invention willbecome apparent from the following description of the invention, whentaken in conjunction with the accompanying drawings, which respectivelyshow:

FIG. 1: a diagram of a transwell chamber used for co-culturing umbilicalcord blood-derived mesenchymal stem cells and neural precursor cells;

FIG. 2: photographs showing the differentiation and proliferation ofNG108-15 (NG108) observed with a phase-contrast microscope (×100), 4 and7 days after culturing NG 108-15 alone or co-culturing therewithumbilical cord blood-derived mesenchymal stem cells, respectively;

FIG. 3: photographs showing the immunostaining result for tubulinbeta-III, an early marker for neuronal differentiation, 7 days afterculturing NG108-15 alone or co-culturing therewith umbilical cordblood-derived mesenchymal stem cells, respectively;

FIG. 4: photographs showing the differentiation and proliferation ofNG108-15 observed with a phase-contrast microscope (×100), 7 days afterculturing the NG108-15 cells alone, supplemented with cAMP orco-culturing with umbilical cord blood-derived mesenchymal stem cellsobtained from two different individuals (hUCB-MSC-1 and hUCB-MSC-2),respectively;

FIG. 5: photographs showing the differentiation and proliferation ofneural stem cells derived from the brain cortex of a fetal mouseobserved with a phase-contrast microscope (×100), 7 days afterco-culturing the neural stem cells with hUCB-MSC-1 and hUCB-MSC-2 ofvarious concentrations, respectively;

FIG. 6: photographs showing the immunostaining result for tubulinbeta-III and microtubule-associated protein 2 (MAP2), early markers ofneuronal differentiation, 7 days after culturing neural stem cellsderived from the brain cortex of the fetal mouse alone or co-culturingtherewith umbilical cord blood-derived mesenchymal stem cells,respectively;

FIG. 7: a graph showing the number of viable cells assessed by thetrypan blue staining, 7 days after co-culturing the NG108-15 withumbilical cord blood-derived mesenchymal stem cells of variousconcentrations; and

FIG. 8: a graph showing the number of viable cells assessed by thetrypan blue staining, 7 days after co-culturing the neural stem cell,derived from the brain cortex of the fetal mouse, with umbilical cordblood-derived mesenchymal stem cells of various concentrations.

DETAILED DESCRIPTION OF THE INVENTION

The inventive composition for inducing differentiation and proliferationof neural precursor cells or neural stem cells to neural cellscharacteristically comprises umbilical cord blood-derived mesenchymalstem cells as an active ingredient.

As used herein, the term “umbilical cord blood” refers to the bloodtaken from the umbilical cord vein which links the placenta of a mammalwith a newborn baby thereof.

The term “umbilical cord blood-derived mesenchymal stem cells” as usedherein refers to mesenchymal stem cells which are isolated from theumbilical cord blood of a mammal, preferably human.

The term “a nerve injury disease” as used herein refers to a diseasethat accompanies, among others, behavior dysfunction due to damagedmotor or sensory nerves. Exemplary nerve injury diseases include Stroke,Parkinson's disease, Alzheimer's disease, Pick's disease, Huntington'sdisease, amyotrophic lateral sclerosis, traumatic central nervous systemdisease and spinal cord injury disease.

The term “treating” refers to improving the condition, alleviatingsymptoms, or delaying the further progress of the disease. The term“prevention” or “inhibition” refer to preventing the manifestation of anot-yet-diagnosed disease or disorder in an animal, preferably a mammal,most preferably human, which is prone to acquire such disease ordisorder; or inhibiting the development of a nerve injury disease.

The term “a neural cell” as used herein refers to a neuron of central orperipheral nervous system, and/or glia such as an astrocyte, anoligodendrocyte and/or a Schwann cell.

In isolating a monocyte comprising mesenchymal stem cells from theumbilical cord blood, a common method such as the Ficoll-Hypaque densitygradient method can be employed. Specifically, said method comprises thesteps of gathering umbilical cord blood from the umbilical vein afterparturition till detachment of placenta; centrifuging the umbilical cordblood with a Ficoll-Hypaque gradient to obtain monocytes; and removingcontaminants therefrom. The obtained monocytes may be subjected toisolation of mesenchymal stem cells therefrom, or to ultrafreezing for along-term safekeeping till use.

The isolation of mesenchymal stem cells from the umbilical cordblood-derived monocytes may be performed by the method of Yang S E etal. (Yang S E et al., Cytotherapy, 6(5):476-486, 2004). Specifically,monocytes are suspended in a medium containing 5 to 30 weight %,preferably 5 to 15 weight % of fetal bovine serum (FBS), the mediumincluding a conventional one such as DMEM, α-DMEM, Eagle's basal mediumor RPMI 1640. Then, the cells in the suspension were divided into mediahaving the same composition as described above and cultured in a 5% CO₂incubator at 37° C. When the cultured cells form a mono-layer,mesenchymal stem cells having a spindle shape are observed. Then, themesenchymal stem cells are subcultured repeatedly until the cells aresufficiently amplified.

According to the present invention, both the differentiation andproliferation of neural precursor cells or neural stem cells to neuralcells can be induced by co-culturing umbilical cord blood-derivedmesenchymal stem cells with neural precursor cells or neural stem cells.Namely, the umbilical cord blood-derived mesenchymal stem cells aresimultaneously effective not only for inducing the differentiation ofthe neural precursor cells or neural stem cells to neural cells, butalso for sustaining and strengthening such effects through increasingthe number of the neural cells, to enhance their therapeutic effects.

Therefore, the present invention provides a use of umbilical cordblood-derived mesenchymal stem cells or a composition comprising thecells for inducing differentiation of neural precursor cells or neuralstem cells to neural cells and proliferation of the resulting neuralcells.

Umbilical cord blood-derived mesenchymal stem cells or a composition ofcomprising the cells can be used for cytotherapy of a patient sufferingfrom a nerve injury diseases, e.g., stroke, Parkinson's disease,Alzheimer's disease, Pick's diseases, Huntington's disease, amyotrophiclateral sclerosis, traumatic central nervous system diseases and spinalcord injury disease, preferably stroke and spinal cord injury disease.

The composition of the present invention may further comprise apharmaceutically acceptable additive.

A pharmaceutical formulation in a unit dosage form may be preparedemploying the composition of the present invention according to theconventional procedures in the art. A formulation for parenteraladministration such as an injection or a topical dosage form ispreferable. The inventive pharmaceutical formulation may further includepharmaceutically-acceptable additives, e.g., fillers, expanders, bindingagents, wetting agents, disintegrants, diluents such as surfactants andother excipients.

The inventive pharmaceutical formulation can be administeredparenterally according to the conventional procedures in the art, forinstance, via direct injection into an injury region as well asinjection into the cerebrospinal fluid, for example, lumbar puncture andparenchymal injection, vein or artery. Preferably, it can beadministered via direct injection into a peripheral or opposite regionof brain or spinal cord injury region. Further, the clinical method ofDouglas Kondziolka (Douglas Kondziolka, Pittsburgh, 1998) may beemployed to administer the inventive pharmaceutical formulations into aninjury region. Specifically, a skull of a subject is incised to make ahole having a diameter of 1 cm and a suspension of a mesenchymal stemcells in HBSS (Hank's balanced salt solution) is injected into the holeby employing a long-needle syringe and a stereotactic frame.

A typical dose of the mesenchymal stem cells may range from 1×10⁵ to1×10⁷ cells/kg body weight/injection, preferably from 5×10⁵ to 5×10⁶cells/kg body weight/injection, which can be administered in a singledose or in divided doses. Further, it should be understood that theamount of the effective ingredient actually administrated to a certainpatient ought to be determined in light of various relevant factorsincluding the amount of neural cells to be differentiated andproliferated, the chosen route of administration, and the body weight,age and sex of an individual patient.

The present invention also provides a method for inducingdifferentiation and proliferation of neural precursor cells or neuralstem cells to neural cells, which comprises co-culturing umbilical cordblood-derived mesenchymal stem cells with the neural precursor cells orthe neural stem cells. The co-culturing may be carried out by mixing theumbilical cord blood-derived mesenchymal stem cells with the neuralprecursor cells or the neural stem cells at a ratio of 1:0.1 to 1:10,preferably, 1:1 to 1:2 based on their cell number and cutting the cellmixture in a conventional cell culture medium such as DMEM, α-DMEM,α-MEM, Eagle's basal medium and RPMI 1640.

The culture medium may further comprise an antibiotic, e.g., gentamicin,and/or 5 to 15 weight % of FBS. The culture period may ranges from 5 to10 days.

The present invention also provides a method for treating a nerve injurydisease which comprises administering the umbilical cord blood-derivedmesenchymal stem cell or a composition comprising the same to the nervecell injury region of a subject in need of treating the nerve injurydisease. The subject may be a mammal including human.

When administered in a therapeutically effective amount, the umbilicalcord blood-derived mesenchymal stem cell induces not onlydifferentiation of neural precursor cells or neural stem cells ofcentral or peripheral nervous system to neural cells, but also theproliferation of the resulting neural cells, thereby resulting in therecovery of the neural functions and treatment of such nerve injurydisease. The term “therapeutically effective amount” may refer to anamount which show treating or preventive effects, and may be exemplifiedby the amount described above. The treating effect of the umbilical cordblood-derived mesenchymal stem cell is greatly enhanced and sustainedfor a long time by its capability of proliferating the regeneratedneural cells.

The following Examples and Test Examples are given for the purpose ofillustration only, and are not intended to limit the scope of theinvention.

Example 1 Isolation and Culture of Umbilical Cord Blood-DerivedMesenchymal Stem Cells (Step 1) Acquisition of Umbilical Cord Blood(UCB)

A UCB sample was obtained from the umbilical vein of a delivering womanunder her consent. Specifically, a 16-gauge needle of a UCB collectionbag containing 44 ml of CPDA-1 anticoagulant (GREEN CROSS) was insertedinto the umbilical vein to allow UCB to flow into the bag. The collectedblood was processed within 48 hours and the cell survival rate was over90%.

(Step 2) Isolation and Amplification of Mesenchymal Stem Cells

The UCB obtained in step 1 was subjected to centrifugation using aFicoll-Hypaque gradient (density: 1.077 g/ml, Sigma) to obtainmonocytes. The monocytes were then washed several times to removeimpurities and suspended in a minimum basal medium containing 5 to 15weight % FBS (HyClone) (α-MEM, Gibco BRL). Subsequently, a pre-measuredamount of the suspension was added to the same media as above andcultured in a 5% CO₂ incubator at 37° C., while exchanging the mediawith a fresh batch of medium twice a week. When the cultured cellsformed a mono-layer, the generation of mesenchymal stem cells having aspindle shape was confirmed with a microscope. The mesenchymal stemcells thus formed were subcultured repeatedly until the cells weresufficiently amplified (Yang S E et al., Cytotherapy, 6(5):476-486,2004).

Example 2 Culture of NG108-15 (NG108)

A mouse brain-derived cell, NG108-15 (Neuroblastoma X glioma hybrid)(ATCC, Cat. No. ATCC-CRL-HB-12317), having similar physiological andmorphological characteristics with a neural precursor cell was culturedin DMEM (Dulbecco's modified Eagle's medium) (4 mM/L glutamine, 4.5 g/Lglucose, 4.0 mg/L pyridoxin-HCl, 0.1 mM hypoxanthine-guanine, 400 nMaminopterin, 0.016 mM thymidine, 5 to 15 weight % FBS).

Example 3 Culture of Neural Stem Cells

Neural stem cells derived from a brain cortex of a fetal mouse(Chemicon, Cat. No. SCR029) were cultured in a neural stem cell basalmedium (20 ng/ml FGF-2, 20 ng/ml EGF and 2 mg/ml heparin).

Example 4 Co-Culture of Umbilical Cord Blood-Derived Mesenchymal StemCells and NG108-15 (I)

The human umbilical cord blood-derived mesenchymal stem cells(hUCB-MSCs) of Example 1 were co-cultured with NG108-15 of Example 2(hUCB-MSCs: NG108-15=1:1) employing a transwell chamber (FIG. 1) and theculture medium of Example 2. As a control group, NG108-15 was culturedalone in the culture medium of Example 2. As shown in FIG. 1, thetranswell chamber was composed of lower and upper compartments whichwere separated from each other by a microporous membrane having 1μm-pores. hUCB-MSCs were placed in the upper compartment, and NG108-15,in the lower compartment.

The differentiation of NG108-15 was observed with a phase-contrastmicroscope (×100) in 4 and 7 days. As shown in FIG. 2, NG108-15co-cultured with hUCB-MSCs differentiated to a form of typical maturedneuron-like cells in which the cells spread long branches anddifferentiated to have a spindle shape.

The differentiated cells were further confirmed to be neuron-like cellsin 7 days employing immunostaining for tubulin-beta III, an early markerof neuronal development. Specifically, hUCB-MSCs, NG 108-15 and amixture thereof were respectively cultured in a cover slide, and blockedby adding them to 10% normal goat serum containing 0.3% triton X-100 for1 hour at a room temperature. The 1^(st) antibody employed in theimmunostaining, an anti-tubulin-beta III mouse monoclonal antibodyconjugated with phycoerythrin (Chemicon), was diluted by one hundredfold with the goat serum and added thereto. The mixture was then keptovernight at 4° C., and the resulting mixture was washed three times(each time for 5 minutes) with 0.01 M PBS.

As shown in FIG. 3, NG108-15 co-cultured with the mesenchymal stem cellsof Example 1 exhibited a distinct response to the immunostaining fortubulin-beta III, verifying that the differentiated were neuron-likecells.

Example 5 Co-Culture of Umbilical Cord Blood-Derived Mesenchymal StemCells and NG108-15 (II)

The human umbilical cord blood-derived mesenchymal stem cells wereobtained by the method of Example 1 from two different individuals(hUCBMSCs-1 and hUCB-MSCs-2). NG108-15 of Example 2 was cultured aloneor co-cultured with hUCB-MSCs-1 or hUCB-MSCs-2 in accordance with themethod of Example 4 for 7 days. Differentiation and proliferation of thecells were observed with a phase-contrast microscope (×100).

As a comparative group, 1 mM of cAMP which induces differentiation ofNG108-15 to neuron-like cells (NeuroReport 9, 1261-1265, 1998) was addedto the culture medium of NG108-15.

As shown in FIG. 4, NG108-15 co-cultured with the mesenchymal stem cellswas differentiated to a form of matured neuron-like cells. There was nosignificant difference between hUCB-MSCs-1 and hUCB-MSCs-2 in theirdifferentiation inducing activities.

Example 6 Co-Culture of Umbilical Cord Blood-Derived Mesenchymal StemCells and Neural Stem Cells (I)

The neural stem cells of Example 3 were cultured alone (control group)or co-cultured with hUCB-MSCs-1 or hUCB-MSCs-2 of Example 5 in theculture medium of Example 3, employing the transwell chamber. hUCB-MSCswere placed in the upper compartment and the neural stem cells, in thelower compartment.

hUCB-MSCs-1 and hUCB-MSCs-2 were respectively added to the medium in theconcentration of 500, 1000, 2000, 4000 and 6000 cells/cm², respectively,and the neural stem cells were co-cultured with the concentration of2000 cells/cm². The differentiation and proliferation of the cells wereobserved with a phase-contrast microscope (×100) (FIG. 5) in 7 days.

As shown in FIG. 5, the neural stem cells co-cultured with themesenchymal stem cells were differentiated to a form of matured neurons.There was no significant difference between hUCB-MSCs-1 and hUCB-MSCs2in their differentiation inducing activities. Further, the extent ofdifferentiation and proliferation of the neural stem cells was directlyproportional to the concentration of the mesenchymal stem cells withwhich co-cultured.

Example 7 Co-Culture of Umbilical Cord Blood-Derived Mesenchymal StemCells and Neural Stem Cells (II)

The neural stem cells of Example 3 were cultured alone (control group)or co-cultured with hUCB-MSCs of Example 1 (hUCB-MSCs: neural stemcells=1:1) in the culture medium of Example 3, employing a transwellchamber. hUCB-MSCs were placed in the upper compartment and the neuralstem cells, in the lower compartment.

Immunostaining was carried out in 4 and 7 days by employing the methodof Example 4 for tubulin-beta III and microtubule-associated protein 2(MAP2), early markers of neuronal development such that it confirmedthat the differentiated were neurons.

As shown in FIG. 6, the neural stem cells co-cultured with themesenchymal stem cells of Example 1 exhibited a distinct response toimmunostaining for tubulin-beta III and MAP2, verifying that thedifferentiated were neurons.

Example 8 Co-Culture of Umbilical Cord Blood-Derived Mesenchymal StemCells with Neural Precursor Cells or Neural Stem Cells

NG108-15 of Example 2 was cultured alone (control group) or co-culturedwith hUCB-MSCs of Example 1, and the neural stem cells of Example 3 wascultured alone (control group) or co-cultured with hUCB-MSCs-1 orhUCB-MSCs-2 of Example 5, according to the methods of Examples 4 and 6,respectively. The number of viable cells was counted in 7 days employingthe trypan blue staining (FIGS. 7 and 8).

As shown in FIGS. 7 and 8, the numbers of NG108-15 and the neural stemcells increased in proportion to the concentration of the mesenchymalstem cells with which co-cultured, implying that the umbilical cordblood-derived mesenchymal stem cells can be simultaneously effective notonly for inducing the differentiation of the neural precursor cells orneural stem cells to neural cells, but also for sustaining andstrengthening such effects through increasing the number of the neuralcells.

While the invention has been described with respect to the abovespecific embodiments, it should be recognized that various modificationsand changes may be made to the invention by those skilled in the artwhich also fall within the scope of the invention as defined by theappended claims.

1-7. (canceled)
 8. A method for treating a nerve injury disease, whichcomprises administering umbilical cord blood-derived mesenchymal stemcells to a nerve cell injury region of a subject in need of treating thenerve injury disease.
 9. The method of claim 8, further comprisesadministering an agent which is selected from the group consisting ofascorbic acid, glia derived neurotrophic factor (GDNF), brain derivedneurotrophic factor (BDNF), retinoic acid, insulin and nerve growthfactor (NGF).
 10. The method of claim 8, wherein the umbilical cordblood-derived mesenchymal stem cells are administered as a culture ofthe umbilical cord blood-derived mesenchymal stem cells and neuralprecursor cells or a culture of the umbilical cord blood-derivedmesenchymal stem cells and neural stem cells.
 11. The method of claim 8,wherein the nerve injury disease is selected from the group consistingof hypoxic ischemic brain injury, stroke, Parkinson's disease,Alzheimer's disease, Pick's disease, Huntington's disease, amyotrophiclateral sclerosis, traumatic central nervous system disease, and spinalcord injury disease.
 12. The method of claim 8, wherein the subject is amammal.